Faculdade de Ciências, BioISI-Biosystems & Integrative Sciences Institute, Universidade de Lisboa, Lisbon, Portugal.
Misión Biológica de Galicia, Sede Santiago de Compostela, Consejo Superior de Investigaciones Científicas (MBG-CSIC), Avda Vigo S/N, 15705, Santiago de Compostela, La Coruña, Spain.
Sci Rep. 2024 Aug 21;14(1):19357. doi: 10.1038/s41598-024-70272-2.
In recent decades an extensive mortality and decline of Quercus suber populations mainly caused by Phytophthora cinnamomi has been observed. In the current study, a chestnut gene homologous to ginkbilobin-2 (Cast_Gnk2-like), which in Ginkgo biloba codifies an antifungal protein, was transferred into cork oak somatic embryos of three different embryogenic lines by Agrobacterium mediated transformation. The transformation efficiency varied on the genotype from 2.5 to 9.2%, and a total of 22 independent transformed lines were obtained. The presence of Cast_Gnk2-like gene in transgenic embryos was verified in all lines by PCR. The number of transgene copies was estimated by qPCR in embryogenic lines with high proliferation ability and it varied between 1 and 5. In addition, the expression levels of Cast_Gnk2-like gene were determined in the embryogenic lines, with higher levels in lines derived from the genotype ALM6-WT. Transgenic plants were obtained from all transgenic lines and evaluated after cold storage of the somatic embryos for 2 months and subsequent transfer to germination medium. In vitro tolerance tests made under controlled conditions and following zoospore treatment showed that plants overexpressing Cast_Gnk2-like gene improved tolerance against Pc when compared to wild type ones.
近几十年来，由于尖孢镰刀菌的影响，栓皮栎种群大量死亡，数量减少。在本研究中，通过根癌农杆菌介导的转化，将栗基因与 ginkbilobin-2（Cast_Gnk2-like）同源物（该基因在银杏中编码一种抗真菌蛋白）导入了三种不同胚性系的栓皮栎体细胞胚中。转化效率因基因型而异，范围为 2.5%至 9.2%，共获得了 22 个独立的转化系。通过 PCR 在所有系中均验证了转基因胚胎中 Cast_Gnk2-like 基因的存在。通过 qPCR 估计了在具有高增殖能力的胚性系中转基因拷贝数，其范围在 1 到 5 之间。此外，在胚性系中还测定了 Cast_Gnk2-like 基因的表达水平，在基因型 ALM6-WT 来源的系中表达水平较高。从所有转化系中均获得了转基因植株，并在体细胞胚经过 2 个月的冷藏和随后转移到萌发培养基后进行了评估。在受控条件下进行的体外耐受力测试以及随后的游动孢子处理表明，与野生型相比，过表达 Cast_Gnk2-like 基因的植株对 Pc 的耐受力得到了提高。
